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Role of Proteolytic Enzymes in Biological Regulation (A Review)*

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Role of Proteolytic Enzymes in Biological Regulation (A Review)* Proc. Natl. Acad. Sci. USA Vol. 73, No. 11, pp. 3825-3832, November 1976 Biochemistry Role of proteolytic enzymes in biological regulation (A Review) * (limited proteolysis/zymogen activation/control mechanisms) HANS NEURATH AND KENNETH A. WALSH Department of Biochemistry, University of Washington, Seattle, Wash. 98195 Contributed by Hans Neurath, September 7, 1976 ABSTRACT Many enzymes, hormones, and other physio- biologically active proteins and probably the first step in protein logically active proteins are synthesized as inactive precursors degradation (12). The specificity of limited proteolysis is best (zymogens) that are subsequently converted to the active form understood in terms of the three-dimensional structure of a by the selective enzymatic cleavage (limited proteolysis) of protein substrate and of the attacking protease because the peptide bonds. The ultimate agency of activating enzymatic containing the susceptible pep- function is limited proteolysis, either in a single activation step region of the protein substrate or in a consecutive series (cascade). The specificity of each ac- tide bond must fit the active site of the attacking protease in tivation reaction is determined by the complementarity of the order for amino acid residues of the substrate to interact with zymogen substrate and the active site of the attacking protease. primary as well as secondary binding sites of the enzyme (13). The sequence of consecutive activation reactions is regulated In general, limited proteolysis is therefore directed toward by the specificity of each enzyme, whereas the degree of am- surface loops and random segments of polypeptide chains rather plification of the initial stimulus is determined by the efficiency sheets. of each activating step. than toward internal domains, helices, or pleated Zymogen activation produces a prompt and irreversible re- The activation of zymogens usually occurs by proteolytic sponse to a physiological stimulus, and is capable of initiating cleavage of a peptide bond in a region that is amino terminal new physiological functions. Typical examples are the processes relative to the active site of the protein. This may be a conse- of blood coagulation, fibrinolysis, complement activation, quence of the process of protein biosynthesis, which proceeds hormone production, metamorphosis, rtilization, supra- in the direction from the amino to the carboxyl end. If it is as- molecular assembly, and digestion. The zymogens of the pan- as creatic serine proteases, in particular, have served as models for sumed that the protein assumes its correct tertiary structure detailed studies of the nature of the molecular changes that are regions of the polypeptide chain are synthesized, the zymogen involved in the dramatic increase in enzymatic activity that will be formed prior to the enzyme. Were the activation peptide ensues upon limited proteolysis of the zymogen. attached to the carboxyl end, trypsin would be synthesized before trypsinogen, fibrin before fibrinogen, or collagen before In recent years, it has become evident that many proteins are procollagen. By synthesizing an inactivating prefix before synthesized as inactive precursors or zymogens and that these synthesizing the active portion of the protein molecule, pre- are subsequently converted to physiologically active forms by mature physiological function is avoided. the selective enzymatic cleavage of peptide bonds. This process The position of zymogen activation in the overall scheme of is known as zymogen activation, a term which initially was physiological control processes is diagrammatically shown in applied to the activation of precursors of proteolytic enzymes Fig. 1. The term zymogen is being used herein to denote in such as trypsinogen, chymotrypsinogen, or procarboxypep- general an inactive precursor that can be converted to an active tidase (1). It is now apparent that the same type of reaction is protein by the cleavage of one or more peptide bonds. This involved in a great variety of biological processes, such as blood process is essentially irreversible because, in common with many coagulation, fibrinolysis, complement reaction, hormone pro- other hydrolytic reactions, proteolysis is an exergonic reaction duction, development, differentiation, and supramolecular under normal physiological conditions and there are no simple assembly, all of which involve zymogen activation in one or biological mechanisms to repair a broken peptide bond. In this more steps (2-9). In the present article, we shall attempt to show respect, zymogen activation differs in kind from the freely that activation by limited proteolysis is indeed an important reversible mechanisms of allosteric transition or covalent control element which can initiate new physiological functions modification (14). Whereas the latter are suited to maintain or or regulate preexistent ones. modulate a steady state of intermediary metabolites, zymogen Virtually all zymogen activation reactions require the en- activation, by virtue of its operational irreversibility, can effect zyme-catalyzed cleavage of a unique peptide bond by "limited unidirectional changes in the cellular environment and can proteolysis." This term was first introduced by Linderstrom- induce new physiological functions. This type of initiation is Lang and Ottesen (10) to describe the restrictive peptide bond more rapid than that regulated by the selective transcription cleavage that induces the conversion of ovalbumin to a different of a genome and is triggered by signals that operate entirely on crystalline form, plakalbumin, under the influence of the the post-translational level. Typical zymogen activation reac- bacterial protease subtilisin. Numerous examples of limited tions are summarized in Table 1. proteolysis have since been described and studied in detail, such In some of these processes, the zymogen is converted to the as the tryptic conversion of chymotrypsinogen to chymotrypsin active protein in a single step, whereas in others the process (1), the release by subtilisin of the amino-terminal segments of involves consecutive steps or cascades (2) which serve to amplify ribonuclease (11), and the conversion of proinsulin to insulin small stimuli to major physiological responses. Many zymogen (5). Limited proteolysis is the last step in the synthesis of many activation reactions may have remained undetected thus far because the precursor becomes activated prior to isolation. * By invitation. From time to time, reviews on scientific and techno- Indeed, isolation procedures are usually designed for maximum logical matters of broad interest are published in the PROCEED- yield of active protein rather than of zymogen and thus may INGS. contravene the demonstration of a zymogen precursor. 3825 Downloaded by guest on September 27, 2021 3826 Biochemistry: Neurath and Walsh Proc. Natl. Acad. Sci. USA 73 (1976) AMINO ACID POOL (b) (o) (b) (C) (d) ZYMOGEN CTIV NACTIVE Limited PROTEIN I I Allosteric PROTEIN Proteolysis Transition or Covalent Modification FIG. 1. Schematic representation of major control mechanisms. (a) Transcription and translation regulate the rate of formation of the various proteins from the amino acid pool. (b) Other controls regulate the rate of degradation of the various proteins to their constitutent amino acids. (c) The activity of the protease that catalyzes zymogen activation may be in turn regulated by a series of consecutive reactions of limited proteolysis (Fig. 2). Activation of a zymogen is essentially irreversible in vivo. (d) Reversible conformational changes are responsive to effector concentrations or to the activities of specific group transferases and hydrolases. CONSECUTIVE ZYMOGEN ACTIVATION trypsinogen by enterokinase is so specific that only a single bond REACTIONS out of 228 in trypsinogen is cleaved and no other protein has A series of consecutive zymogen activation reactions is shown yet been reported to be a substrate for enterokinase (16). Thus, diagrammatically in Fig. 2. X, Y, and Z are zymogens, each the site of generation of active trypsin is restricted to the con- having the potential of being converted to an active protein. fluence of these two secretory streams (Fig. 3). Active trypsin Conversion of the zymogen X to the protease Xa is triggered by in turn catalyzes the conversion of other pancreatic zymogens a specific physiological stimulus; in the ensuing cascade, the to their active forms, i.e., the chymotrypsinogens, proelastase, product of one reaction is a catalyst for the next. The sequence the procarboxypeptidases, and prophospholipase. This system of the events is determined by the specificity of each enzyme constitutes a two-stage cascade. and the degree of amplification of the initial stimulus is de- A more complex and extensive cascade system is found in the termined by the efficiency of each activating step. For instance, blood coagulation process, shown in Fig. 4. In fact, the term one molecule of Xa might produce 103 molecules of Ya, which cascade or waterfall was introduced by Macfarlane (17) and in turn produce 106 molecules of active protein. When the zy- by Davie and Ratnoff in 1964 (18) in connection with this series mogen is produced by one cell type and the activating protease of reactions. Five known proteolytic reactions occur along the by another, communication between the two cell types adds so-called intrinsic pathway, which is mediated entirely by another element to the control mechanism (15). For instance, components found
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